Project Summary Bacterial resistance to antibiotics is a serious and growing problem, and the development of new antibiotics is critical in the fight against antibiotic resistance. One important class of antibiotics are polyketide compounds, which are produced by modular polyketide synthase enzymes. I will identify novel polyketide antibiotics by generating a polyketide biosensor and by applying large-scale mutagenesis, thereby working towards a general strategy to identify novel biosynthetic products. An E. coli biosensor will be constructed by first fusing a ligand-binding domain that binds to diverse polyketide compounds to required bacterial transcriptional component. The hybrid protein will then be engineered for ligand-dependent stability, such that it is stabilized in the presence of ligand and destabilized in the absence of ligand. In this design, increased biosensor stability is linked to increased reporter gene transcription, enabling the rapid measurement of ligand levels. In parallel, gene synthesis methods will be used to construct a synthetic polyketide pathway, incorporating restriction sites in key locations within the relevant genes. This synthetic pathway will be used to construct chimeric pathways in which acyltransferase domains from other polyketide synthase pathways are substituted for the native domain. Acyltransferase domains control which new substrates are incorporated into the growing polyketide backbone, and substitution of acyltransferase domains with unique substrate tolerance will enable the biosynthesis of diverse polyketide compounds. These chimeric pathways will be evaluated for production of their polyketide product. Finally, large-scale mutagenesis and biosensor-based selection will be used to rescue the biosynthesis activity of poorly performing chimeric pathways to produce novel polyketide antibiotics. This strategy of biosensor-based selection coupled with large-scale mutagenesis can be broadly applied to other biosynthetic pathways to produce novel compounds.